Use of a protein or gene regulating color change in petunia hybrida in regulating color change in petunia hybrida and breeding method

Abstract

This invention discloses the application and cultivation method of a protein or gene that regulates petunia flower color change, belonging to the field of plant genetic engineering technology. This invention clones a gene that regulates petunia flower color change from petunia. PH7 It belongs to the ERD6L family of proteins. It was obtained using the CRISPR / Cas9 gene editing system. PH7 Gene knockout lines showed no significant difference in petal color from the wild type in the early stages of flower development and at the beginning of flowering. However, 2-3 days after flowering, blue patches began to appear on the petals of the knockout lines, gradually turning completely blue as the flower development progressed. This indicates that... PH7 Genes play a crucial role in regulating the formation of petunia color-changing flowers. This invention provides new insights into the molecular mechanisms regulating petunia color-changing flower formation and offers new targets for the breeding of new petunia color-changing flower varieties.

Description

Technical Field

[0001] This invention relates to the application and cultivation method of a protein or gene that regulates the color change of petunias, belonging to the field of plant genetic engineering technology. Background Technology

[0002] Flower color, as one of the most intuitive ornamental traits of garden plants, directly determines their landscape application value and market economic value. Breeding new varieties of "color-changing flowers" that exhibit dynamic color changes during a single flowering period or across different flowering periods is an important breeding direction to break through existing limitations in flower color and greatly enrich the color expression and artistic effects of ornamental plants. Petunia hybrida, as one of the most important flowerbed and potted ornamental plants globally, occupies a core position in landscaping and home gardening due to its wide flower spectrum, abundant flowering, and long flowering period. However, stable natural color-changing flower germplasm resources for petunias are lacking in nature, and the physiological and biochemical pathways and molecular regulatory networks involved in flower color variation are extremely complex, leading to slow progress in elucidating the molecular mechanisms of petunia color-changing and in targeted breeding. Therefore, systematically discovering and functionally analyzing the key genes regulating the dynamic changes in petunia flower color and elucidating their mechanisms of action has crucial theoretical and applied value for promoting molecular breeding of color-changing ornamental plants.

[0003] In recent years, with the development of plant mutant libraries and high-throughput sequencing technologies, research on color-changing flowers has made some progress in some species. For example, in hydrangeas, changes in the pH value of petal cell vacuoles are the main reason for the change in flower color with soil acidity and alkalinity; while in morning glories, changes in the expression of genes related to anthocyanin modification have been found to cause flower color change with the opening time. These studies reveal the complexity of how the internal physiological environment of petals (such as pH value and metal ion concentration) and anthocyanin metabolic pathways jointly regulate flower color variation. Nevertheless, key transporter protein genes that can directly regulate endogenous physiological indicators in petal cells and lead to stable, heritable color-changing traits have not yet been fully explored and utilized.

[0004] Petunias possess abundant transposon insertion mutant resources, providing ideal material for functional gene cloning. Petunias have an extremely active transposon mutant library. Utilizing this resource, researchers have for the first time screened and obtained two petunia mutant materials with stable color-changing phenotypes, named ph7. m / m and ph7 - / -The mutant flowers exhibit a significant color change from red to blue / purple during opening, providing valuable genetic material for studying the mechanism of color-changing flowers. Preliminary studies on the ph7 mutant revealed that: 1) the pH value of its petal tissue significantly increases as the flower opens, which may be one of the key factors driving the anthocyanin color change; 2) by comparing transcriptome and hybrid offspring segregation ratios, the key gene PH7 controlling this trait was identified, and its encoded product is homologous to the Arabidopsis glucose / proton cotransporter AtERDL6, belonging to the early response dehydration (ERD6-like) subfamily of monosaccharide transporters; 3) the dynamic changes in glucose content in the petals of the ph7 mutant are closely related to the color change process.

[0005] ERD6-like (ERDL) proteins, an important subfamily of monosaccharide transporters, are named after the fact that the first member was discovered in Arabidopsis thaliana in response to drought stress. Current research generally suggests that members of this family are primarily involved in the transmembrane transport of sugars in plants and play a key role in the accumulation of soluble sugars. For example, in Arabidopsis, AtERD6L6 is located in the vacuolar membrane, and its loss of function leads to impaired glucose transport from the vacuolar, resulting in increased intracellular glucose levels (Klemens et al. (2014) Overexpression of a proton-coupled vacuolar glucose exporter impairs freezing tolerance and seed germination. New Phytologist 202, 188-197; Poschetetal. (2011) A novel Arabidopsis vacuolar glucose exporter is involved in cellular sugar homeostasis and affects the composition of seed storage compounds. Plantphysiology 157, 1664-1676.). In grapes, VvERD6L13 is located in the plasma membrane and has a specific transport capacity for sucrose, enabling the transport of soluble sugars from the apoplast to grape fruit cells (Breia et al. (2020) VvERD6L13 is a grapevine sucrose transporter highly up-regulated in response to infection). (by Botrytiscinerea and Erysiphe necator. Plant Physiology and Biochemistry 154, 508-516.). In citrus, the expression level of CsERD6L is positively correlated with sugar accumulation in the fruit and can continuously and stably mediate the transport of glucose from vacuoles to the cytoplasm (Zheng et al. (2014) Isolation, phylogenetic relationship and expression profiling of sugar transporter genes in sweet orange (Citrus sinensis). Plant Cell 119, 609-624.).In watermelon, the ERD6L protein can transport hexoses such as glucose from the vacuoles of the fruit to the cytoplasm, thereby affecting the quality and taste of the watermelon fruit (Renetal. (2023) An update on sugar allocation and accumulation in fruits. Plant Physiology 193, 888-899.). In apples, MdERD6L-1, as a proton cotransporter on the vacuolar membrane, can transport glucose from the vacuolar to the cytoplasm. Overexpression of MdERD6L-1 causes a large influx of glucose into the cytoplasm, thereby inducing the expression of sugar transporter genes MdTST1 and MdTST2, which further leads to an increase in glucose, fructose and sucrose content in the vacuolar (Zhu et al. (2021) MdERDL6-mediated glucose efflux to the cytosol promotes sugar accumulation in the vacuole through up-regulating TSTs in apple and tomato. Proceedings of the National Academy of Sciences 118, e2022788118; Zhu et al. (2023) The SnRK2.3-AREB1-TST1 / 2 cascade activated by cytosolic glucoseregulates sugar accumulation across tonoplasts in apple and tomato. Nature Plants 9, 951–964.). These studies consistently demonstrate that the ERDL protein family primarily functions in regulating the transport and accumulation of soluble sugars.

[0006] However, to date, all research on ERDL proteins has been limited to their regulation of sugar transport and non-flower color-related agronomic traits (such as fruit sugar content). No published literature or patents have reported that this family of proteins can directly or indirectly regulate petal color. Particularly in ornamental plants, the molecular mechanism by which ERDL proteins coordinate sugar metabolism, cell pH, and anthocyanin stability to ultimately lead to visible dynamic changes in flower color remains completely unknown, representing a gap in this technological field.

[0007] In summary, current understanding of the formation mechanism of color-changing flowers in petunias and other ornamental plants is still limited, and key target genes that can be effectively used for molecular breeding are lacking. Although the functions of the ERDL protein family in sugar transport have been extensively elucidated, their novel functions in flower color formation and dynamic changes have never been revealed. Therefore, in-depth analysis of how the petunia PH7 gene precisely regulates flower color change by integrating sugar signals and cellular pH environment can not only fill the knowledge gap in the theory of color-changing flower regulation, but also provide key genes and innovative technical solutions with independent intellectual property rights for the molecular design and targeted breeding of new ornamental plant color-changing flower varieties, which has significant scientific value and broad industrial application prospects. Summary of the Invention

[0008] The identification and screening of the PH7 gene were based on forward genetics. By screening a library of petunia transposon insertion mutants, we discovered two color-changing flower mutants for the first time. Our research revealed that this color-changing phenotype was caused by changes in pH levels within the petal cells, so we named this allele PH7 (our research group had previously identified six other flower color-related alleles, named PH1-PH6). Later, by performing transcriptome sequencing on the two ph7 mutants and wild-type petals, combined with Denovo transcriptome assembly, flower color phenotype, and genotype association analysis, we finally confirmed that the color-changing flowers in the two ph7 mutants were caused by mutations in the ERD6L6 gene induced by transposon insertion. Subsequently, using CRISPR / Cas9 gene editing technology, we successfully edited the PH7 (ERD6L6) gene, obtaining a color-changing flower phenotype consistent with the transposon insertion mutants, further demonstrating that the PH7 (ERD6L6) gene is a key gene regulating the formation of petunia color-changing flowers.

[0009] Therefore, this invention cloned a gene, PH7, belonging to the ERD6L family of proteins, from petunias to regulate flower color change. Using the CRISPR / Cas9 gene editing system, the obtained PH7 knockout lines showed no significant difference in petal color compared to the wild type in the early stages of flower development and at the initial opening. However, 2-3 days after opening, blue patches began to appear on the petals of the knockout lines, gradually turning almost entirely blue as the flower development progressed. This indicates that the PH7 gene plays a crucial role in regulating the formation of color-changing flowers in petunias. This invention provides new insights into the molecular mechanisms regulating color-changing flower formation and offers new targets for breeding new varieties of petunias and other ornamental plants with color-changing flowers.

[0010] This invention first provides the application of a protein or its encoding gene that regulates the color change of petunia flowers in regulating the color change of petunia flowers;

[0011] The amino acid sequence of the protein is shown in SEQ ID No. 1.

[0012] Specifically, the nucleotide sequence of the encoding gene is shown in SEQ ID No. 2.

[0013] More specifically, genetic engineering techniques are used to knock out or weaken the gene encoding petunia flower color change through editing systems, or to weaken or lose the function of the protein that regulates petunia flower color change.

[0014] Preferably, the gene encoding the color change of petunia flowers is knocked out using CRISPR / Cas9 gene editing.

[0015] This invention also provides a method for cultivating petunia plants or varieties that change color, which uses genetic engineering techniques to knock out or weaken the coding gene for petunia flower color change in petunias through an editing system, or weaken or lose the function of the protein that regulates petunia flower color change.

[0016] The amino acid sequence of the protein is shown in SEQ ID No. 1.

[0017] Specifically, the nucleotide sequence of the encoding gene is shown in SEQ ID No. 2.

[0018] More specifically, the gene encoding the color change of petunia flowers was knocked out using CRISPR / Cas9 gene editing.

[0019] Optionally, it also includes steps for further screening and breeding to obtain petunia varieties that change color.

[0020] The technical solution provided by this invention can be used to cultivate new varieties of petunia with color-changing flowers, and has practical value. Attached Figure Description

[0021] Figure 1 Identification of the flower color phenotype of the ph7 mutant petunia. A) shows the petal color phenotype of the ph7 mutant; b) shows the determination of anthocyanin content and components in the petals of the ph7 mutant and wild-type by HPLC-MS; c) shows the pH measurement of the crude extracts of the petals of the ph7 mutant and wild-type. The R176 strain is wild-type, while the R173 and R146 strains are stable ph7 mutants (ph7...). - / - ) and transposon insertion unstable mutant (ph7) m / m The black arrow indicates a typical dTph1 transposon revertant sector. DAO: Days after flowering.

[0022] Figure 2 Preliminary identification of the PH7 gene. Among them, 'a' represents the unstable mutant allele ph7. R146The first exon contains an insertion of the dTph1 transposon (gray triangle, 289 bp); the stable mutant allele is ph7. R173 An 8bp insertion (blue italicized) was found in the fourth exon; b shows the ph7 transposon insertion mutation gene detected by PCR in the F2 generation of R146 and R176 hybrid plants. R146 The red, orange, and purple dots in the image indicate the PH7 genotype, which is inferred from the plant's flower color phenotype. The presence of only a ~800bp band in the electrophoresis image indicates that the plant's genotype is PH7. m / m The presence of both ~800bp and ~500bp bands indicates that the plant's genotype is ph7. + / m The presence of only a ~500bp band indicates that the plant's genotype is PH7. + / + Tn represents the transposon.

[0023] Figure 3 Phylogenetic analysis of the PH7 protein.

[0024] Figure 4 The effect of exogenous glucose solution application on the color change of ph7 transposon insertion mutant.

[0025] Figure 5 The expression patterns of the PH7 gene in different tissues of petunia, where PS: petal stage; DAO: number of days after flowering.

[0026] Figure 6 Subcellular localization of PH7 in petunia petal protoplasts. RFP-SYP122 labeled cell membranes; blue indicates anthocyanin autofluorescence; scale bar = 10 μm.

[0027] Figure 7 PhPH7 gene CRIPSR / Cas editing plasmid map. a, target site construction plasmid map designed by exon1 and intron1; b, target site construction plasmid map designed by exon3 and exon6.

[0028] Figure 8 Petunia PH7 gene knockout mutant (ph7 - / - The flower color gradually turns blue as the flower opens. a) Knockout mutant line 1 has a 1bp insertion in the third exon of the PH7 gene; b) Knockout mutant line 2 has a 14bp deletion in the sixth exon of the PH7 gene. DAO: Days after flowering. Detailed Implementation

[0029] This invention is illustrated through specific embodiments in order to better understand the invention, but does not constitute a limitation thereof.

[0030] Example 1

[0031] (1) Identification of flower color phenotype of ph7 mutant and screening of ph7 gene. By screening the petunia transposon insertion mutant library, two petunia color-changing flower mutants were obtained for the first time and named ph7. m / m (R146) and ph7 - / - (R173). To clarify the mechanism of color-changing flower formation, anthocyanins in the petals of mutant and wild-type plants were detected by HPLC-MS; and the pH values ​​of crude extracts from the petals of mutant and wild-type plants were determined. To screen for genes at the PH7 locus, ph7... m / m (R146) and ph7 - / - Transcriptome sequencing (RNA-seq) was performed on petals of the (R173) mutant and wild type at different developmental stages. The PH7 gene was screened and preliminarily identified by comparing the transcriptome and the segregation ratio of the hybrid offspring.

[0032]

[0033] The amino acid sequence of the protein encoded by the PH7 gene is: MSFREENNSNEEGGRGDLRKPFLHTGSWYRMGSAQTSSMLGSSQAFRDSSISVLACVMIVALGPIQFGFTSGYSSPTQTAITKDLKLTVSEFSLFGSLSNVGAMVGAISSGQIAEYIGRKGSLMIAAIPNIIGWLSISFAKDLSFLYMGRLLEGFGVGIISYTVPVYIAEIAPQNLRGALGSVNQLSVTIGIFLAYLLGFFVNWRVLAVLGTLPCLALIPGLFFIPESPRWLAKMGLTEDF ETSLQVLRGFEADISIEVNEIKRSVASTSRKSAIRFADLKQRRYWLPLMIGGLLVLQQLGGTNGVIFYSSNIFLSAGISSSNAATFGVGAIQVVATGVATWLVDKTGRRLLLIVSSCGMTVSLLIVS IAFFLKGFVAEDSTLYGALGILSVVGVVLMIIAFSLGMGPIPWLIMSEILPVKIKGLAGSVATLANWFCSWVITATAPLLLAWSSGGTFALYTIVCAFTVAFVTIWVPETKGKTLEEIQFSFR (SEQID No.1).

[0034] (3) Spatiotemporal expression analysis of PH7 gene. Samples of different tissues and organs (roots, stems, leaves) and different stages of flower development (bud stage (PS1-5), anther closure stage (PS6), and 1-5 days after flowering (DAO1-5)) of wild-type petunias (M1×V30) were collected and sent to Biomarker for transcriptome sequencing. After the transcriptome data were analyzed using standard procedures, the spatiotemporal expression pattern of PH7 gene was analyzed.

[0035] (4) Subcellular localization of PH7 in petunia petal cells. A GFP and PH7 fusion protein vector (p35S:GFP-PH7) was constructed and, together with the p35S:RFP-SYP122 plasmid (the fusion protein is located in the vacuolar membrane), was transiently transfected into wild-type (M1×V30) petal protoplasts via PEG-mediated transfection. After 24 hours of transformation, the fluorescent protein signal was observed using an LSM710 laser confocal microscope to determine the subcellular localization of PH7 protein in petunia cells.

[0036] (5) Obtaining the PH7 gene editing mutant. Using the CRISPR-GE online tool developed by Academician Liu Yaoguang of South China Agricultural University (http: / / skl.scau.edu.cn / ), four editing target sites of the PH7 gene were designed based on the genomic DNA sequence of the PH7 gene (Table 1). The target sites were combined in pairs and the target gRNA expression cassette was ligated into the pYLCRISPR / Cas9-MTdi vector using the Golden Gate cloning method (for details of the method, see Ma et al. (2015) A Robust CRISPR / Cas9 System for Convenient High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Molecular Plant, 8, 1274-1284). The plasmid map is shown in [link to plasmid map]. Figure 7 , Figure 7 In the image, 'a' represents the plasmid map constructed at two target sites designed for exon1 and intron1 of the PH7 gene. Figure 7 Figure b shows the plasmid maps constructed at two target sites designed at exon3 and exon6 of the PH7 gene. The two PH7 gene editing vectors were transformed into Agrobacterium AGL0, and then into the petunia M1×V30 line using the leaf disc method. Positive plants were selected by kanamycin resistance and detected by PCR. Primers were designed upstream and downstream of the target sites, and the sequences containing the target sites in the positive transformed plants were amplified by PCR. Homozygous PH7 gene knockout mutants were screened by sequencing, and flower color phenotypes were observed.

[0037]

[0038] Table 1. PH7 gene editing target site sequences

[0039] .

[0040] Example 2

[0041] (1) Identification of flower color phenotype in ph7 mutants. ph7 m / m Transposon insertion mutant (R146), ph7 - / - Stable mutants (R173) and PH7 + / + The wild-type petals are red for two days after flowering, while the petals of the ph7 mutant gradually turn purple / blue seven days after flowering. Figure 1 (a) To preliminarily determine the influencing factors of the gradual bluening of the flower color in the ph7 mutant, samples of ph7 were extracted. - / - Anthocyanins in petals of the stable mutant 2 days (red) and 7 days (blue / purple) after flowering were analyzed by HPLC-MS, with anthocyanins in the corresponding wild-type petals as a control. - / - The composition and content of anthocyanins (mainly cyanidin) in the mutant were not significantly different from those in the wild type. Figure 1 (b) Further measurements of pH 7. - / - The pH of crude petal extracts from stable mutants 7 days after flowering and wild-types at the corresponding time was found to be significantly higher in the petals of the pH7 mutant. Figure 1 (c). Based on the above results, it can be inferred that the blue coloration of the petals of the ph7 mutant is not caused by changes in anthocyanin composition or content, but may be caused by a gradual increase in the pH of the vacuoles in the petal epidermal cells.

[0042] (2) Screening and preliminary identification of the PH7 gene. Through screening and preliminary identification of the PH7 gene... m / m Transposon insertion mutant (R146), ph7 - / - Stable mutants (R173) and PH7 + / + Transcriptome sequencing was performed on petals of wild-type (R176) flowers before and 5 days after flowering. A candidate gene, PH7, was successfully screened using a combination of bioinformatics methods. Figure 2 As shown in Figure a, ph7 R146 The allele contains a dTph1 transposon insertion in the first exon, while ph7... R173 The allele contains an 8bp insertion in the fourth exon. The F1 generation (ph7) was obtained by crossing R146 and R176. + / m The F1 generation was obtained through self-pollination (V2009 and V2011 lines). Based on the flower color phenotype of the F2 generation, the genotype of PH7 in each line was inferred (e.g., Figure 2(As shown by the red, orange, and purple dots in b); upstream and downstream primers were designed based on the PH7 gene transposon insertion site. Using extracted gDNA from V2009 and V2011 plants as templates, PCR was used to detect the PH7 gene transposon insertion. Figure 2 As shown in Figure b, the genotype of PH7 in the F2 generation plants identified by PCR results is consistent with the PH7 genotype inferred from the flower color phenotype, thus preliminarily confirming the correctness of the screened PH7 gene.

[0043] Phylogenetic trees were constructed using the amino acid sequences of monosaccharide transporter family members from Arabidopsis thaliana and Petunia spp., and the results are as follows: Figure 3 As shown, petunia PH7 is closely related to Arabidopsis thaliana AtERD6-like4 and AtERD6-like6, belonging to the ERD6-like protein subfamily.

[0044] (3) Expression of PH7 in different tissues of petunia and subcellular localization of its encoded protein. Transcriptome analysis of different tissues of wild-type petunia (M1×V30) revealed ( Figure 4 The PH7 gene was expressed in different petunia tissues, but the expression levels varied, with the lowest expression in the anthers. The expression was relatively low during the petunia bud stage (PS1-5), rose to the highest during the flowering stage (PS6), and gradually decreased with the number of days after flowering (DAO1-5), suggesting that it may play an important role in the physiological processes after flowering.

[0045] By constructing the fusion protein expression vector GFP-PH7 and transiently expressing it in petunia petal protoplasts, it was found that the fusion protein GFP-PH7 was localized on the vacuolar membrane in both petal epidermal cells and mesophyll cells. Figure 5 These results suggest that petunia PH7 may also play a role in the transport of glucose from vacuoles to the cytoplasm in petal cells.

[0046] (4) Petunia PH7 may be involved in glucose transport in petal cells. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) m / m Transposons were inserted into newly opened flowers of mutants (petals open, anthers closed, petals red) and then into H2O and 2% glucose solutions respectively. The differences in flower color phenotypes were observed under the same conditions. Figure 6 As shown, feeding with a 2% glucose solution can significantly reduce pH 7. m / m The degree to which the mutant petals turned blue / purple initially proved that: 1) the formation of the discolored flowers of the ph7 mutant was caused by changes in the glucose content of the petals; 2) ph7 may be involved in glucose transport in petal cells.

[0047] (5) Petal phenotype of PH7 gene knockout mutant. To further verify the correctness of the PH7 gene screening, two CRISPR / Cas9 gene editing vectors for the PH7 gene were constructed ( Figure 7 (a and b), and successfully edited the PH7 gene in the petunia line M1×V30, obtaining two knockout mutant lines ph7. - / - In knockout mutant line 1, the PH7 gene contains a 1bp insertion in the third exon. Figure 8 (a), while in knockout mutant line 2, the PH7 gene contains a 14bp sequence deletion in the sixth exon (a). Figure 8 (b) In addition, such as Figure 8 As shown in Figures a and b, the petal color of flowers from knockout mutants 1 and 2 changes from red to blue as the flowers open, and their flower color phenotype is consistent with that of the ph7 transposon insertion mutant and the stable mutant. Figure 1 This further proves that the screened PH7 gene is a key gene regulating the formation of color-changing flowers.

Claims

1. The application of a protein or its encoding gene that regulates the color change of petunias in regulating the color change of petunias, wherein the encoding gene is knocked out or the protein expressing it that regulates the color change of petunias is rendered ineffective by using a genetic engineering method through an editing system, and the amino acid sequence of the protein is shown in SEQ ID No.

1.

2. The application as described in claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID No.

2.

3. The application as described in claim 1, characterized in that, The coding gene was knocked out using CRISPR / Cas9 gene editing techniques.

4. A method for cultivating color-changing petunia plants or varieties, characterized in that, Genetic engineering techniques were used to knock out the coding gene that regulates the color change of petunias through an editing system, or to cause the protein that regulates the color change of petunias to lose its function. The amino acid sequence of the protein is shown in SEQ ID No.

1.

5. The method as described in claim 4, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID No.

2.

6. The method as described in claim 4, characterized in that, The gene encoding the color change of petunia flowers was knocked out using CRISPR / Cas9 gene editing.

7. The method according to any one of claims 4 to 6, characterized in that, It also includes steps for further screening and breeding to obtain petunia varieties that change color.

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